U.S. patent application number 10/006002 was filed with the patent office on 2003-06-19 for reducing myelin-mediated inhibition of axon regeneration.
Invention is credited to He, Zhigang, Kim, Jieun A., Koprivica, Vuk, Wang, Kevin C..
Application Number | 20030113325 10/006002 |
Document ID | / |
Family ID | 21718786 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030113325 |
Kind Code |
A1 |
He, Zhigang ; et
al. |
June 19, 2003 |
Reducing myelin-mediated inhibition of axon regeneration
Abstract
Oligodendrocyte-myelin glycoprotein (OMgp)-specific binding
agents are used to reduce OMgp-mediated axon growth inhibition.
Mixtures of axons and OMgp and mixtures of Nogo receptor (NgR) and
OMgp are used in pharmaceutical screens to characterize agents as
inhibiting binding of NgR to OMgp and promoting axon
regeneration.
Inventors: |
He, Zhigang; (Boston,
MA) ; Wang, Kevin C.; (Boston, MA) ;
Koprivica, Vuk; (Boston, MA) ; Kim, Jieun A.;
(Boston, MA) |
Correspondence
Address: |
RICHARD ARON OSMAN
SCIENCE AND TECHNOLOGY LAW GROUP
75 DENISE DRIVE
HILLSBOROUGH
CA
94010
|
Family ID: |
21718786 |
Appl. No.: |
10/006002 |
Filed: |
December 3, 2001 |
Current U.S.
Class: |
424/146.1 ;
435/7.2 |
Current CPC
Class: |
G01N 2500/02 20130101;
C07K 2317/55 20130101; G01N 33/6896 20130101; A61K 38/1787
20130101; C07K 16/18 20130101; A61K 2039/505 20130101 |
Class at
Publication: |
424/146.1 ;
435/7.2 |
International
Class: |
A61K 039/395; G01N
033/53; G01N 033/567 |
Goverment Interests
[0001] This work was supported by Federal Grant No. 1R21NS41999-01
from NINDS. The government may have rights in any patent issuing on
this application.
Claims
What is claimed is:
1. A method for reducing axon growth inhibition mediated by
oligodendrocyte-myelin glycoprotein (OMgp) and detecting resultant
reduced axon growth inhibition, the method comprising steps:
contacting a mixture comprising an axon and isolated OMgp with an
agent and under conditions wherein but for the presence of the
agent, the axon is subject to growth inhibition mediated by the
OMgp; and detecting resultant reduced axon growth inhibition.
2. A method according to claim 1, wherein the isolated OMgp
consists essentially of OMgp.
3. A method according to claim 1, wherein the isolated OMgp
consists essentially of OMgp, wherein the OMgp is soluble and
GPI-cleaved.
4. A method according to claim 1, wherein the OMgp is recombinantly
expressed on a surface of a cell.
5. A method according to claim 1, wherein the mixture is in
vitro.
6. A method according to claim 1, wherein the agent is a candidate
agent not previously characterized to bind OMgp nor reduce axon
growth inhibition mediated by OMgp and the detecting step
characterizes the candidate agent as reducing axon growth
inhibition mediated by OMgp.
7. A method according to claim 1, wherein the agent is a candidate
agent not previously characterized to reduce axon growth inhibition
mediated by OMgp and the detecting step characterizes the candidate
agent as reducing axon growth inhibition mediated by OMgp.
8. A method according to claim 1, wherein the agent comprises an
OMgp-specific antibody fragment.
9. A method according to claim 1, wherein the agent is soluble Nogo
receptor (NgR) peptide sufficient to specifically bind the OMgp and
competitively inhibit binding of the OMgp to NgR
10. A method for reducing axon growth inhibition mediated by OMgp
and detecting resultant reduced axon growth inhibition, the method
comprising steps: contacting a mixture comprising an axon and OMgp
with an exogenous OMgp-specific binding agent and under conditions
wherein the agent binds the OMgp and but for the presence of the
agent, the axon is subject to growth inhibition mediated by the
OMgp, and detecting resultant reduced axon growth inhibition.
11. A method according to claim 10, wherein the mixture is in
vitro.
12. A method according to claim 10, wherein the agent is a
candidate agent not previously characterized to bind OMgp nor
reduce axon growth inhibition mediated by OMgp and the detecting
step characterizes the candidate agent as reducing axon growth
inhibition mediated by OMgp.
13. A method according to claim 10, wherein the agent is a
candidate agent not previously characterized to reduce axon growth
inhibition mediated by OMgp and the detecting step characterizes
the candidate agent as reducing axon growth inhibition mediated by
OMgp.
14. A method according to claim 10, wherein the agent comprises an
OMgp-specific antibody fragment.
15. A method according to claim 10, wherein the agent is soluble
NgR peptide sufficient to specifically bind the OMgp and
competitively inhibit binding of the OMgp to NgR.
16. A method according to claim 10, wherein the agent is soluble
NgR peptide sufficient to specifically bind the OMgp and
competitively inhibit binding of the OMgp to NgR, wherein the
peptide consists essentially of a sequence within SEQ ID NO:1 at
least six residues in length.
17. A method for characterizing an agent as inhibiting binding of
NgR to OMgp, the method comprising the steps of: incubating a
mixture comprising NgR, OMgp and an agent under conditions whereby
but for the presence of the agent, the NgR and OMgp exhibit a
control binding; and detecting a reduced binding of the NgR to the
OMgp, indicating that the agent inhibits binding of the NgR to the
OMgp.
18. A method according to claim 17, wherein at least one of the NgR
and OMgp is soluble and GPI-cleaved.
19. A method according to claim 17, wherein one of the NgR and OMgp
is soluble and GPI-cleaved and the other is membrane-bound.
20. A method according to claim 17, wherein at least one of the NgR
and OMgp is recombinantly expressed on a surface of a cell.
Description
INTRODUCTION
[0002] 1. Field of the Invention
[0003] The invention is in the field of reducing meylin-mediated
inhibition of axon regeneration.
[0004] 2. Background of the Invention
[0005] Most axons in the adult mammalian central nervous system
(CNS) have little innate ability for repair after injury (Horner
and Gage, 2000). Examination of post-lesioned axons in the adult
nervous system reveals that their proximal ends are able to form
growth cones, the primary navigating entity of growing axons, that
appear both morphologically and functionally identical to those of
developing nerve fibers (reviewed by Tessier-Lavgine and Goodman,
2000). Studies over the past two decades have identified a number
of guidance cues that can influence the motility and directionality
of projecting axons during embryonic development (Tessier-Lavigne
and Goodman, 1996; Song and Poo, 2001). It is believed that the
combined influences of attractants and repellents orchestrate the
precise motile behavior of individual axons. Conversely, either a
lack of permissive cues and/or the presence of dominant inhibitors
in the adult CNS seem to contribute significantly to the inability
of lesioned axons to regenerate (Schwab & Bartholdi, 1996;
Fournier and Strittmatter, 2001).
[0006] In addition to the actual physical barrier presented by
glial scarring at the lesion sites (KcKeon et al., 1991; Davies et
al., 1997; Moon et al., 2001), inhibitory factors in
oligodendrocyte-derived myelin clearly play a role in limiting axon
regeneration. Immobilized CNS myelin proteins have been shown to
potently inhibit axon outgrowth from a variety of neurons in vitro
(Schwab and Caroni, 1988; Savio and Schwab, 1989). In addition,
anti-myelin antibodies have been used to neutralize the inhibitory
effects of myelin and, more importantly, stimulate regeneration of
the corticospinal tract in vivo (Schnell and Schwab, 1990; Bregmann
et al., 1995; Huang et al., 1999). Most of the efforts towards
identifying these myelin-associated inhibitors thus far have
centered on assaying biochemical fractions of CNS myelin for
growth-inhibitory activity in vitro and then isolating the
corresponding molecules (Caroni and Schwab, 1988; McKerracher et
al., 1994; Spillmann et al., 1998; Niederost et al., 1999). Several
myelin components have been identified as putative inhibitors of
regeneration through such approaches. One such component is myelin
associated glycoprotein (MAG), a transmembrane protein with a five
immunoglobulin domain-harboring extracellular region (Arquint et
al., 1987; Salzer et al., 1987). Even though MAG is capable of
inhibiting axon outgrowth from different types of cultured neurons
(McKerracher et al., 1994; Mukhopadhyay et al., 1994; Li et al.,
1996; Tang et al., 1997), knockout animals provide conflicting data
on the effects of removing the MAG protein product on axon
regeneration in vivo (Bartsch et al., 1995; Li et al., 1996;
Schafer et al., 1996). In addition to MAG, neurite
outgrowth-inhibitory activity has also been found to associate with
chondroitin sulfate proteoglycans (CSPGs) in CNS myelin (Niederost
et al., 1999). However, it is unclear whether this inhibitory
activity results from CSPGs themselves or from a combination with
additional factors.
[0007] In addition to MAG and CSPGs, another putative inhibitor,
Nogo/NI-250 (Caroni and Schwab, 1988), has attracted much attention
because an anti-NI-250 monoclonal antibody, IN-1, had been shown to
neutralize the growth-inhibitory effects of myelin-associated
inhibitors both in vitro and in vivo. Remarkably, IN-1 treatment
resulted in long-distance fiber growth and increased axonal
sprouting within the adult CNS (Schnell and Schwab, 1990; Bregmann
et al., 1995; Thallmair et al., 1998). The partial-peptide sequence
of biochemically purified NI-250 (Spillmann et al., 2000) allowed
several groups to clone the corresponding cDNA which encodes three
Nogo isoforms, designated Nogo-A (presumably NI-250), -B, and -C,
presumably generated from alternative splicing or differential
promoter usage (Chen et al., 2000; GrandPre et al., 2000; Prinjha
et al., 2000). Surprisingly, Nogo-A, or Reticulon 4-A, appears to
be a member of the reticulon protein family, and associates
primarily with the endoplasmic reticulum (ER) (GrandPre et al.,
2000). Nogo-A protein is believed to contain at least two
transmembrane domains. Interestingly, both the amino-terminal
cytoplasmic (amino-Nogo) (Chen et al., 2000; Prinjha et al., 2000;
Fournier et al., 2001) and the lumenal/extracellular (Nogo-66)
(GrandPre et al., 2000; Fournier et al., 2001) domains of Nogo are
able to inhibit axon growth in vitro. Insights into the signaling
mechanism(s) that mediate the inhibitory activity of Nogo came with
the recent identification of a functional receptor for Nogo-66 by
expression cloning (Fournier et al., 2001). The Nogo-66 receptor
(NgR), a protein which associates with the plasma membrane through
a glycosylphosphatidylinositol (GPI) anchor, is expressed in most
postnatal neuronal populations and binds Nogo-66 with high affinity
(Fournier et al., 2001). Furthermore, early embryonic chick retinal
ganglion cells that are normally insensitive to Nogo-66 become
responsive upon expression of NgR (Fournier et al., 2001),
suggesting that NgR is able to mediate the activity of Nogo-66.
However, it is unclear how the primarily intracellularly localized
Nogo protein reaches and acts on regenerating axons.
[0008] Previous biochemical studies have demonstrated the presence
of additional inhibitory activities of unknown molecular identity
(McKerracher et al., 1994; Spillmann et al., 1999; Niederost et
al., 1999). As most myelin proteins are assumed to be associated
with the plasma membrane of axon-ensheathing oligodendrocytes, we
reasoned that these proteins could either be in the transmembrane
form or be tethered to the membrane with covalent linkers. As both
MAG and Nogo are transmembrane proteins, and the
glycosylphosphatidylinositol (GPI)-mediated covalent linkage
appears to be the most common structural feature of many
membrane-associated axon guidance molecules (for example, Ranscht
and Dours-Zimmermann, 1991; Xu et al., 1998; Nakashiba et al.,
1999; O'Leary and Wilkinson, 1999), we decided to investigate the
possibility that GPI-linked CNS myelin proteins may play a role in
inhibiting axon regeneration. By utilizing
phosphatidylinositol-specific phospholipase C (PI-PLC) to release
GPI-linked proteins from CNS myelin, we found these proteins to
have a potent growth cone-collapsing activity. We found that
oligodendrocyte-myelin glycoprotein (OMgp), a previously identified
GPI-linked CNS myelin protein with unknown function (Mikol and
Stefansson, 1988; Habib et al., 1998a; Habib et al. 1998b),
provides such inhibitory activity. Furthermore, through the use of
both loss- and gain-of-function experiments, we demonstrate that
OMgp acts through NgR to inhibit axon regeneration.
SUMMARY OF THE INVENTION
[0009] The invention provides methods and compositions for reducing
OMgp-mediated axon growth inhibition. In one embodiment, the method
comprising steps (a) contacting a mixture comprising an axon and
isolated OMgp with an agent and under conditions wherein but for
the presence of the agent, the axon is subject to growth inhibition
mediated by the OMgp; and (b) detecting resultant reduced axon
growth inhibition. In an alternative embodiment, the method
comprises steps: (a) contacting a mixture comprising an axon and
OMgp with an exogenous OMgp-specific binding agent and under
conditions wherein but for the presence of the agent, the axon is
subject to growth inhibition mediated by the OMgp, whereby the
agent binds the OMgp and reduces the growth inhibition; and (b)
detecting resultant reduced axon growth inhibition.
[0010] These methods may be practiced with isolated neurons in
vitro, or with neurons in situ. Suitable agents include (i) a
candidate agent not previously characterized to bind OMgp nor
reduce axon growth inhibition mediated by OMgp; (ii) a candidate
agent not previously characterized to reduce axon growth inhibition
mediated by OMgp; (iii) an OMgp-specific antibody fragment; (iv) a
soluble NgR peptide sufficient to specifically bind the OMgp and
competitively inhibit binding of the OMgp to NgR; etc. In more
particular embodiments, the recited isolated OMgp consists
essentially of OMgp, particularly wherein the OMgp is soluble and
GPI-cleaved and/or the OMgp is recombinantly expressed on a surface
of a cell.
[0011] The invention also provides methods and compositions for
characterizing an agent as inhibiting binding of NgR to OMgp. In
one embodiment, this method comprising the steps (a) incubating a
mixture comprising NgR, OMgp and an agent under conditions whereby
but for the presence of the agent, the NgR and OMgp exhibit a
control binding; and (b) detecting a reduced binding of the NgR to
the OMgp, indicating that the agent inhibits binding of the NgR to
the OMgp.
[0012] The method may be practiced in a variety of alternative
embodiments, such as (i) wherein at least one of the NgR and OMgp
is soluble and GPI-cleaved; (ii) wherein one of the NgR and OMgp is
soluble and GPI-cleaved and the other is membrane-bound; (iii)
wherein at least one of the NgR and OMgp is recombinantly expressed
on a surface of a cell; etc.
[0013] The invention also provides compositions and mixtures
specifically tailored for practicing the subject methods. For
example, an in vitro mixture for use in the subject binding assays
comprises NgR, OMgp and an agent, wherein at least one of the NgR
and OMgp is soluble and GPI-cleaved. Kits for practicing the
disclosed methods may also comprise printed or electronic
instructions describing the applicable subject method.
DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
[0014] The following descriptions of particular embodiments and
examples are offered by way of illustration and not by way of
limitation. Unless contraindicated or noted otherwise, in these
descriptions and throughout this specification, the terms "a" and
"an" mean one or more, the term "or" means and/or and
polynucleotide sequences are understood to encompass opposite
strands as well as alternative backbones described herein.
[0015] In one embodiment, the invention provides a method for
reducing axon growth inhibition mediated by OMgp and detecting
resultant reduced axon growth inhibition, the method comprising
steps: contacting a mixture comprising an axon and isolated OMgp
with an agent and under conditions wherein but for the presence of
the agent, the axon is subject to growth inhibition mediated by the
OMgp; and detecting resultant reduced axon growth inhibition,
indicating that the agent reduces axon growth inhibition mediated
by OMgp.
[0016] The recited axons are mammalian neuron axons, preferably
adult neural axons, which may be peripheral or, preferably CNS
neuron axons. As exemplified below, the method may be applied to
neural axons in vitro or in situ.
[0017] OMgp is a natural, mammalian CNS myelin glycoprotein (see,
Habib et al. 1998a, 1998b) which functions as a ligand of the Nogo
Receptor (NgR) on CNS axons. OMgp cDNA has been cloned from several
species, including human (Genbank Accn No. NM.sub.--002544), mouse
(Genbank Accn No. NM.sub.--019409), and cow (Genbank Accn No.
S45673). Note that OMgp cDNA encodes two alternative initiating
methionine residues; compare, Genbank Accession Nos. M63623 (human)
and S67043 (mouse). OMgp may be membrane-bound through a GPI
linkage or cleaved therefrom. As exemplified herein, OMgp may be
obtained on or cleaved from naturally expressing myelin. Also as
exemplified herein, OMgp may also be expressed recombinantly in
suitable recombinant expression systems, wherein functional
expression may be confirmed by the growth cone collapsing assays
described herein.
[0018] The recited isolated OMgp is provided isolated from other
components of OMgp's natural myelin mileau, which may be effected
by purification from such components or expression of the OMgp in a
non-natural system. In particular embodiments, the isolated OMgp is
accompanied by other components which provide or interfere with or
alter the axon growth inhibitory or NgR binding activity of the
OMgp. Preferred isolated OMgp is purified or recombinantly
expressed, particularly on a surface of a cell.
[0019] The recited agent may be characterized as an OMgp-specific
binding agent or, particularly as applied to pharmaceutical
screens, an agent not previously characterized to bind OMgp nor
reduce axon growth inhibition mediated by OMgp, wherein the agent
is a candidate agent and the detecting step characterizes the
candidate agent as reducing axon growth inhibition mediated by
OMgp. Similarly, the agent may be a candidate agent not previously
characterized to reduce axon growth inhibition mediated by OMgp,
wherein the detecting step characterizes the candidate agent as
reducing axon growth inhibition mediated by OMgp.
[0020] Detailed protocols for implementing the recited steps are
exemplified below and/or otherwise known in the art as guided by
the present disclosure. The recited contacting and detecting steps
are tailored to the selected system. In vitro systems provide ready
access to the recited mixture using routine laboratory methods,
whereas in vivo systems, such as intact organisms or regions
thereof, typically require surgical or pharmacological methods.
More detailed such protocols are described below. Similarly, the
detecting step is effected by evaluating different metrics,
depending on the selected system. For in vitro binding assays,
these include conventional solid-phase labeled protein binding
assays, such as ELISA-type formats, solution-phase binding assays,
such as fluorescent polarization or NMR-based assays, etc. For
cell-based or in situ assays, metrics typically involve assays of
axon growth as evaluated by linear measure, density, host mobility
or other function improvement, etc.
[0021] In another embodiment, the invention provides a method for
reducing axon growth inhibition mediated by OMgp and detecting
resultant reduced axon growth inhibition by (a) contacting a
mixture comprising an axon and OMgp with an exogenous OMgp-specific
binding agent and under conditions wherein the agent binds the OMgp
and but for the presence of the agent, the axon is subject to
growth inhibition mediated by the OMgp, and (b) detecting resultant
reduced axon growth inhibition.
[0022] This protocol may similarly be practiced with in vitro or in
vivo, particularly in situ, mixtures. Note that in this embodiment,
the agent is necessarily an exogenous OMgp-specific binding agent
and the recited OMgp need not be isolated, i.e. it may be present
in the context of its native myelin. Accordingly, this aspect of
the invention provides methods for reducing axon growth inhibition
mediated by OMgp in its native mileau. By reducing axon growth
inhibition, the methods assist the repair of axons following injury
or trauma, such as spinal cord injury. In addition, the methods may
be applied to alleviate dysfunction of the nervous system due to
hypertrophy of neurons or their axonal projections, such as occurs
in diabetic neuropathy.
[0023] An OMgp-specific binding agent exogenous to an axon or
mixture comprising an axon is not naturally present with the axon
or mixture. The OMgp-specific binding agents specifically bind the
OMgp of the recited mixture and thereby functionally inhibit the
axon collapse and/or NgR binding mediated by the OMgp. We have
exemplified suitable OMgp binding agents from diverse structures.
Initial agents were identified by selecting high affinity OMgp
binders from natural NgR peptides. These assays identified a number
of OMgp-specific NgR peptides encompassing NgR LLR sequences,
including the exemplified species: hNR260/308, mNR260/308 and
rNR260/308. Natural OMgp-specific NgR peptide sequences were
subject to directed combinatorial mutation and binding analysis.
Resultant synthetic-sequence OMgp-specific peptides include the
exemplified species: s1NGR260/308, s2NR260/308 and s3NR260/308. We
also used a variety of OMgp peptide immunogens to generate
OMgp-specific antibodies and antibody fragments, including the
exemplified monoclonal antibodies OM-H2276 and OM-H5831 and the
exemplified fragments OMF-H7712 and OMF-H6290. OMgp-specific
binding agents are also found in compound libraries, including the
exemplified commercial fungal extract and a synthetic combinatorial
organo-pharmacophore-biased libraries. Structural characterization
of the exemplified OMgp binding agents (XR-178892, XR-397344,
XR-573632, SY-73273M, SY-32340L and SY-95734E) is effected by
conventional organic analysis.
[0024] Of particular interest are size-minimized NgR LLR peptides
which effectively compete for OMgp ligand binding. We synthesized
and screened large libraries of NgR peptides for their ability to
bind OMgp and thereby reduce OMgp-mediated axon growth inhibition.
This work identified numerous competitive binding peptides of
varying length within a 49 amino acid region of a NgR C-terminal
leucine rich repeat, exemplified with human, mouse and rat repeat
sequences (hNR260/308, SEQ ID NO:1; mNR260/308, SEQ ID NO:2; and
rNR260/308, SEQ ID NO: 3). Competitive peptides demonstrating
>20% competitive activity compared with the source 49 mer are
subject to combinatorial mutagenesis to generate synthetic peptide
libraries from which we screen for even higher affinity binders.
Preferred competitive peptides consist, or consist essentially of a
size-minimized sequence within the disclosed human source 49 mer,
preferably a sequence of fewer than 48, 38, 28 or 18 residues,
wherein at least 6, 8, 12 or 16 residues are usually required for
specific binding. Obtaining additional such native sequence and
synthetic competitive peptides involves only routine peptide
synthesis and screening in the disclosed binding and growth
assays.
[0025] In particular applications, the target cells are injured
mammalian neurons in situ, e.g. Schulz M K, et al., Exp Neurol.
February 1998; 149(2): 390-397; Guest J D, et al., J Neurosci Res.
Dec. 1, 1997; 50(5): 888-905; Schwab M E, et al., Spinal Cord. July
1997; 35(7): 469-473; Tatagiba M, et al., Neurosurg March 1997;
40(3): 541-546; and Examples, below. For these in situ
applications, compositions comprising the OMgp binding agent may be
administered by any effective route compatible with therapeutic
activity of the compositions and patient tolerance. For example,
for CNS administration, a variety of techniques is available for
promoting transfer of therapeutic agents across the blood brain
barrier including disruption by surgery or injection, drugs which
transiently open adhesion contact between CNS vasculature
endothelial cells, and compounds which facilitate translocation
through such cells. The compositions may also be amenable to direct
injection or infusion, intraocular administration, or within/on
implants e.g. fibers such as collagen fibers, in osmotic pumps,
grafts comprising appropriately transformed cells, etc.
[0026] In a particular embodiment, the binding agent is delivered
locally and its distribution is restricted. For example, a
particular method of administration involves coating, embedding or
derivatizing fibers, such as collagen fibers, protein polymers,
etc. with therapeutic agents, see also Otto et al. (1989) J
Neurosci Res. 22, 83-91 and Otto and Unsicker (1990) J Neurosc 10,
1912-1921. The amount of binding agent administered depends on the
agent, formulation, route of administration, etc. and is generally
empirically determined and variations will necessarily occur
depending on the target, the host, and the route of administration,
etc.
[0027] The compositions may be advantageously used in conjunction
with other neurogenic agents, neurotrophic factors, growth factors,
anti-inflammatories, antibiotics etc.; and mixtures thereof, see
e.g. Goodman & Gilman's The Pharmacological Basis of
Therapeutics, 9.sup.th Ed., 1996, McGraw-Hill. Exemplary such other
therapeutic agents include neuroactive agents such as in Table
1.
1TABLE 1 Neuroactive agents which may be used in conjunction with
OMgp binding agents. NGF Heregulin Laminin NT3 IL-3 Vitronectin
BDNF IL-6 Thrombospondin NT4/5 IL-7 Merosin CNTF Neuregulin
Tenascin GDNF EGF Fibronectin HGF TGFa F-spondin bFGF TGFb1
Netrin-1 LIF TGFb2 Netrin-2 IGF-I PDGF BB Semaphorin-III IGH-II
PDGF AA L1-Fc Neurturin BMP2 NCAM-Fc Percephin BMP7/OP1 KAL-1
Abbreviations: NGF, nerve growth factor; NT, neurotrophin; BDNF,
brain-derived neurotrophic factor; CNTF, ciliary neurotrophic
factor; GDNF, glial-derived neurotrophic factor; HGF, hepatocyte
growth factor; FGF, fibroblast growth factor; LIF, leukemia
inhibitory factor; IGF, insulin-like growth factor; IL,
interleukin; EGF, epidermal growth factor; TGF, transforming growth
factor; PDGF, platelet-derived growth factor; BMP, bone morphogenic
protein; NCAM, neural cell adhesion molecule.
[0028] In particular embodiments, the OMgp binding agent is
administered in combination with a pharmaceutically acceptable
excipient such as sterile saline or other medium, gelatin, an oil,
etc. to form pharmaceutically acceptable compositions. The
compositions and/or compounds may be administered alone or in
combination with any convenient carrier, diluent, etc. and such
administration may be provided in single or multiple dosages.
Useful carriers include solid, semi-solid or liquid media including
water and non-toxic organic solvents. As such the compositions, in
pharmaceutically acceptable dosage units or in bulk, may be
incorporated into a wide variety of containers, which may be
appropriately labeled with a disclosed use application. Dosage
units may be included in a variety of containers including
capsules, pills, etc.
[0029] The invention also provides pharmaceutical screens for
inhibitors of OMgp-NgR binding, particularly, methods for
characterizing an agent as inhibiting binding of NgR to OMgp by:
(a) incubating a mixture comprising NgR, OMgp and an agent under
conditions whereby but for the presence of the agent, the NgR and
OMgp exhibit a control binding; and (b) detecting a reduced binding
of the NgR to the OMgp, indicating that the agent inhibits binding
of the NgR to the OMgp.
[0030] NgR is a natural, mammalian neural axon protein (Fournier et
al., 2001, Nature 409, 341-46) which functions as a receptor for
Nogo66 and for OMgp. NgR cDNA has been cloned from several species,
including human (Genbank Accn No. BC011787), mouse (Genbank Accn
No. NM-022982), and rat (Genbank Accn No. AY028438). NgR may be
membrane-bound through a GPI linkage or cleaved therefrom. As
exemplified herein, NgR may be obtained on or cleaved from
naturally expressing myelin. Also as exemplified herein, NgR may
also be expressed recombinantly in suitable recombinant expression
systems, wherein functional expression may be confirmed by the
growth cone collapsing assays described herein.
[0031] The screening method is amenable to a wide variety of
different protocols. For example, in particular embodiments, at
least one of the NgR and OMgp is soluble and GPI-cleaved, one of
the NgR and OMgp is soluble and GPI-cleaved and the other is
membrane-bound, and at least one of the NgR and OMgp is
recombinantly expressed on a surface of a cell.
[0032] The invention also provides compositions and mixtures
specifically tailored for practicing the subject methods. For
example, an in vitro mixture for use in the subject binding assays
comprises premeasured, discrete and contained amounts of NgR, OMgp
and an agent, wherein at least one of the NgR and OMgp is soluble
and GPI-cleaved. Kits for practicing the disclosed methods may also
comprises printed or electronic instructions describing the
applicable subject method.
EXAMPLES
[0033] Identification of OMgp as an inhibitor of axon outgrowth. To
examine whether any GPI-linked proteins in CNS myelin may act as
inhibitors of axon regeneration, we treated purified bovine white
matter myelin with phosphatidylinositol-specific phospholipase C
(PI-PLC). PI-PLC cleaves GPI anchors at the junction between the
bilayer-associated diacylglycerol and the peptide-associated
phosphoinositol ring, resulting in the release of the polypeptide
chain with its attached anchor glycan from the lipid bilayer (Low,
1987). The PI-PLC-released proteins were then examined for their
ability to alter growth cone morphology in a growth cone collapse
assay using embryonic day 13 (E13) chick dorsal root ganglia
(DRG)(Luo et al., 1993; He and Tessier-Lavigne, 1997; Fournier et
al., 2001). Our data show that PI-PLC-released CNS myelin proteins,
when added to the DRG culture medium, exhibited potent growth cone
collapsing activity.
[0034] To further characterize the inhibitory activity in the
PI-PLC-released proteins, we analyzed solubilized proteins by
SDS-PAGE and silver staining and found that a band of approximately
110 kDa in size was significantly enriched in this fraction.
Previous studies have identified several GPI-linked proteins in CNS
myelin, including the 120 kDa N-CAM120 (Bhat and Silberberg, 1986),
the 62-70 kDa 5'-nucleotidase (Cammer et al., 1985; Zimmermann et
al., 1992), the 135 kDa F-3 (Koch et al., 1997), 90 and 120 kDa
brevican (Seidenbecher et al., 1995), and the 110 kDa
oligodendrocyte-myelin glycoprotein (OMgp, Mikol and Stefansson et
al., 1988). Since OMgp was the closest in size to our 110-kDa band,
we used anti-OMgp antibodies to detect the enrichment of cleaved
OMgp in the PI-PLC supernatants by Western blot. Our data show that
anti-OMgp antibodies detected a band of similar size in the
PI-PLC-treated supernatants, indicating that OMgp is one of the
components released from CNS myelin by PI-PLC.
[0035] Next, we examined whether purified recombinant OMgp protein
was able to influence the axon outgrowth behavior of cultured
neurons. We engineered a construct that drives the expression of a
polyhistidine-tagged mouse OMgp protein in COS-7 cells so that the
expressed OMgp-His proteins could be readily purified by a
nickle-agarose column. Similar to the PI-PLC-treated myelin
supernatants, purified OMgp-His protein, but not proteins purified
from control vector-transfected COS-7 cells, induced the collapse
of growth cones derived from E13 chick DRG neurons. To further
demonstrate that the OMgp protein acts as an inhibitor of axon
regeneration, we assessed its ability to affect axon outgrowth in
cultured neurons. Our data show that OMgp-His inhibited axon
outgrowth of cerebellar granule neurons from postnatal day 7-9
(P7-9) rats when the protein was provided in either immobilized or
soluble form. The OMgp-induced inhibitory responses were strikingly
reminiscent of that brought about by treatment of the neurons with
an alkaline phosphatase-fusion protein containing the 66 amino acid
lamenal/extracellular domain of Nogo-A (AP-66). Comparable effects
were also observed in neurite outgrowth assays with differentiated
PC12 cells. Together, these results indicate that OMgp is a novel
inhibitor of axon regeneration.
[0036] Expression cloning of the Nogo-66 receptor as an
OMgp-binding protein. To further investigate the mechanisms by
which OMgp inhibits axon outgrowth, we utilized an expression
cloning strategy to isolate OMgp-binding proteins. The coding
region of OMgp was fused to that of alkaline phosphatase (AP), a
readily detectable histochemical reporter (Flanagan and Leder,
1990; Flanagan and Cheng, 2000), and of a polyhistidine tag for
expression and subsequent purification of the chimeric protein in
COS-7 cells. The purified protein appeared as a major band of about
180 kDa as detected by Western blotting, consistent with the
combined sizes of OMgp and AP; a few smaller peptides, apparently
degradation products, were also detected in the preparation. The
AP-OMgp protein, but not AP alone, bound to axons when applied to
the culture medium of P9 granule cells.
[0037] We next took advantage of AP-OMgp binding to identify cell
surface OMgp-binding proteins by expression cloning (He &
Tessier-Lavigne, 1997; Flanagan and Cheng, 2000; Fournier et al.,
2001). Pools of a complementary DNA expression library from adult
human brain, representing approximately 250,000 independent clones,
were transfected into COS-7 cells and screened for the presence of
cells that bound AP-OMgp. As expected, un-transfected COS-7 cells
did not bind AP-OMgp, but transfection with two pools of 5,000
clones each resulted in a few OMgp-binding cells. After screening
several rounds of subpools, we isolated two cDNAs that encoded the
OMgp-binding proteins. Sequencing analysis revealed that both cDNAs
contained the full-length coding region of the nogo-66 receptor
(NgR), which had been previously identified as a high-affinity
receptor for the lamenal/extracellular domain of Nogo (Fournier et
al., 2001). Upon transfection of the NgR cDNA into neuroblastoma
N2A cells, we were able to determine the binding affinity of
expressed NgR for OMgp as 5 nM, similar to what had been determined
for Nogo-66 (7 nM, Fournier et al., 2001). These data indicate that
NgR is a high-affinity OMgp-binding protein. We next performed
coimmunoprecipitation experiments by incubating GST or a GST-NgR
fusion protein containing the entire extracellular domain of NgR
(GST-NgR) with AP or AP-OMgp. Our data show that GST-NgR, but not
control GST protein, bound selectively to AP-OMgp, indicating a
direct interaction between OMgp and NgR.
[0038] OMgp binds the Nogo-66 receptor through its leucine-rich
repeat domain. Amino acid sequence analysis indicated that OMgp,
like NgR (Fournier et al., 2001), is a GPI-linked protein
containing a leucine-rich-repeat (LRR) domain, which has been
implicated in mediating different protein-protein interactions
(Kobe and Deisenhofer, 1994). In addition, OMgp was predicted to
have a C-terminal domain with serine-threonine repeats (Mikol et
al., 1990). To determine which region(s) of OMgp is responsible for
its binding to the NgR, we generated two additional constructs
fusing AP to the N- or C-terminal region of OMgp and used the
conditioned medium from COS-7 cells transfected with each of these
constructs to test for NgR binding. When comparable amounts of each
AP fusion protein were incubated with either control or
NgR-expressing CHO cells, AP-OMgp-LRR showed strong binding to
NgR-expressing cells, indicating that the LRR domain of OMgp might
mediate the interaction between OMgp and NgR. It remains to be
determined whether this interaction is mediated by the LRR domains
of both proteins or whether OMgp binding to NgR occurs independent
of the NgR LRR domain. We also observed weak binding of the AP
fusion protein containing only the serine-threonine repeats of OMgp
(AP-OM-S/T) to NgR expressing cells. As this domain of OMgp has
been proposed to harbor the attachment sites of O-linked
carbohydrates (Mikol et al., 1990), we repeated the binding assays
in the presence of heparin, a non-specific binding competitor, and
found that the AP-OM-S/T and NgR interaction was not affected.
[0039] PI-PLC treatment abolishes neuronal responses to OMgp. To
assess whether NgR mediates the axon outgrowth-inhibitory effects
of OMgp, we took advantage of the fact that the GPI-linked NgR
protein can be released by PI-PLC and examined whether PI-PLC
treatment could affect the axon responsiveness to OMgp. Consistent
with a previous study (Fournier et al., 2001), PI-PLC treatment did
not alter the growth cone morphology of E13 chick DRG neurons, but
rendered these axons insensitive to Nogo-66. Similarly, PI-PLC
treatment also abolished the growth cone-collapse activity of OMgp.
However, the growth cone collapse activity of Semaphorin 3A (Sema
3A), known to be mediated by transmembrane receptor molecules
including neuropilin-1 and members of the plexin family (reviewed
by Raper et al., 2000), was not affected by PI-PLC treatment.
[0040] Nogo-receptor confers neuronal responsiveness to OMgp. To
assess whether NgR is capable of mediating OMgp-induced inhibitory
activity on axon outgrowth, we took a gain-of-function approach to
examine whether expression of NgR was able to confer
OMgp-responsiveness to otherwise insensitive neurons. It has been
shown previously that chick E7 retinal ganglion neurons are
insensitive to Nogo-66, but that expression of NgR in these neurons
rendered their growth cones to be responsive to Nogo-66 (Fournier
et al., 2001). Using the same strategy, we made a recombinant
herpes simplex virus (HSV) that drives expression of a flag-tagged
full-length NgR in infected neurons. Upon infection, most of the E7
retinal ganglion axons expressed the NgR protein as assessed by
immunostaining with an anti-flag antibody. No significant
morphological alterations were observed in the HSV-infected
neurons. Consistent with a previous study (Fournier et al., 2001),
expression of flag-NgR conferred a Nogo-66-induced growth cone
collapse response to E7 retinal ganglion cells. Furthermore, the
growth cones of NgR-expressing axons also became collapsible by
OMgp. In contrast, a control virus driving the expression of LacZ
did not alter the axonal responses of the same neurons to either
Nogo-66 or OMgp.
[0041] To further substantiate the ability of NgR to mediate
OMgp-elicited neuronal responses, we screened a number of neuronal
cell lines and found that mouse neuroblastoma cells (N2A) are
insensitive to both OMgp and Nogo-66. These cells consistently
failed to bind the AP-OMgp protein. In order to extend our
findings, we established a N2A cell line that stably expresses
flag-NgR, and found that neurite outgrowth in these cells was
dramatically inhibited when challenged with both soluble and
immobilized OMgp-His. The same observations were made in parallel
with soluble and immobilized AP-66. Taken together, these results
indicate that NgR mediates the axon outgrowth inhibitory activity
of OMgp.
[0042] Purification and PI-PLC treatment of myelin. Myelin was
prepared from white matter of bovine brain according to established
protocols (Norton and Poduslo, 1973). In brief, white matter
tissues were homogenized in 0.32 M sucrose in phosphate-buffered
saline (PBS) and the crude myelin that banded at the interphase of
a discontinuous sucrose gradient (0.32M/0.85 M) was collected and
purified by two rounds of osmotic shock with distilled water and
re-isolation over the sucrose gradient. For PI-PLC treatment,
aliquots of myelin suspension in water (10 mg/ml) were incubated
with or without 2.5 U/ml PI-PLC (Sigma) at 37.degree. C. for 2 hr,
prior to centrifugation (360,000 g for 60 min). The supernatants
were concentrated, partitioned in Triton X-114, and used for assays
and detection with Western analysis.
[0043] Expression cloning and binding experiments. Sequences
encoding mouse OMgp were amplified from Marathon-ready mouse cDNA
(Clontech) and confirmed by sequencing analysis, prior to
subcloning into the expression vector AP-5 (Flanagan and Cheng,
2000) for expressing an AP-OMgp fusion protein tagged with both a
polyhistidine and a myc epitope. The resultant plasmid DNA was
transfected into COS-7 cells and the secreted protein purified
using nickel-agarose resins (Qiagen).
[0044] Cell surface binding and expression cloning were performed
as described previously (He and Tessier-Lavigne, 1997). To detect
AP-OMgp binding, cultures were washed with binding buffer (Hanks
balanced salt solution containing 20 mM Hepes, pH 7.5, and 1 mg/ml
bovine serum albumin (BSA)). The plates were then incubated with
AP-OMgp-containing binding buffer for 75 min at room temperature.
After extensive washing and heat inactivation, bound AP fusion
proteins were detected by AP staining using NBT and BCIP as
substrate. For saturation analysis, we disrupted cells and detected
bound AP fusion proteins using r-nitrophenyl phosphate as
substrate.
[0045] For expression cloning of OMgp-binding proteins, pools of
5,000 arrayed clones from a human brain cDNA library (Origene
Technologies, Rockville, Md.) were transfected into COS-7 cells,
and AP-OMgp binding was assessed. We isolated single NgR cDNA
clones by sub-dividing the pools and sequencing analysis.
[0046] Generation of recombinant proteins and virus,
immunoprecipitation, and Western analysis. To express recombinant
OMgp for function assays, we subcloned the coding region sequence
of mouse OMgp (amino acids 23-392) into pSecTag B to express
his-tagged OMgp protein (OM-His) in COS-7 cells. The expressed
OMgp-His protein was purified using a nickel resin. To construct
recombinant herpes simplex viruses (HSV), cDNAs for flag-tagged NgR
or LacZ were inserted into the HSV amplicon HSV-PrpUC and packaged
into the virus using the helper 5dl1.2, as described previously
(Neve et al., 1997). The resultant viruses were purified on a
sucrose gradient, pelleted, and resuspended in 10% sucrose. The
titer of the viral stocks was .about.4.0.times.10.sup.7 infectious
units/ml. For each study, aliquots from the same batches of the
viral vectors were used. In order to produce recombinant Nogo-66
protein, the sequence of Nogo-66 was amplified from a human cDNA
clone, KIAA0886, from the Kazusa DNA Research Institute and used to
generate a construct for expressing AP-66 protein as described by
GrandPre et al (2000). The production of Sema3A, co-precipitation
and Western analysis were described previously (He &
Tessier-Lavigne, 1997).
[0047] Growth cone collapse assays. Chick E13 dorsal root ganglion
(DRG) and E7 retina were isolated and cultured as described
previously (Luo et al., 1993; He and Tessier-Lavigne, 1997;
Fournier et al., 2001). Overnight cultured DRG explants were used
for growth cone collapse assays. To assess the effects of PI-PLC
treatment, cultures were pre-incubated with 2 U/ml PI-PLC for 30
min prior to treatment with individual test proteins for an
additional 30 min. To express NgR in E7 retinal ganglion neurons,
we infected the explants for 24 hr. Some cultures infected with
flag-NgR or LacZ were processed for cytohistochemical staining to
verify protein expression. After incubation with each test protein
for 30 min, retinal explants were fixed in 4% paraformaldehyde and
15% sucrose, followed by staining with rhodamine-conjugated
phalloidin.
[0048] Neurite outgrowth assay. P7-9 rat cerebellar neurons were
dissected and cultured as described previously (Huang et al.,
1999). In brief, 96-well plates were first coated with solubilized
nitrocellulose and preincubated with 5 mg/ml poly-D-lysine (Sigma).
Purified proteins in a 2-ml drop volume were placed in the center
of these wells and incubated for 4 hr at 37.degree. C. Cerebellar
neurons were then plated at a density of 1.times.10.sup.5 cells per
well. The cells were cultured for 24 hr prior to fixation with 4%
parafonnaldehyde and staining with an anti-b-tubulin antibody (TuJ,
Covance).
[0049] Exemplary OMgp Binding Agents. An AP-OMgp fusion protein,
prepared as described above, was used to evaluate the OMgp binding
affinity of a variety of candidate binding agents. The selected
binding assay formats are guided by structural requirements of the
candidate agents and include COS-expression, solid phase ELISA-type
assay, and fluorescent polarization assays. Candidate agents were
selected from natural and synthetic peptide libraries biased to
natural NgR LRR (supra) sequences, OMgp-specific monoclonal
antibody (Mab) and Mab fragment libraries, a commercial fungal
extract library, and a synthetic combinatorial
organo-pharmacophore-biased library. Selected exemplary high
affinity OMgp-specific binding agents subject to in vivo activity
assays (below) are shown in Table 2.
2TABLE 2 Selected exemplary high-affinity OMgp-specific binding
agents; (u), structure not yet determined. Sequence/ Binding OMgp
Binding Agent Class/Source Structure Assay 1. hNR260/308 natural
peptide SEQ ID NO:1 + + + + 2. mNR260/308 natural peptide SEQ ID
NO:2 + + + + 3. rNR260/308 natural peptide SEQ ID NO:3 + + + + 4.
s1NR260/308 synthetic peptide SEQ ID NO:4 + + + + 5. s2NR260/308
synthetic peptide SEQ ID NO:5 + + + + 6. s3NR260/308 synthetic
peptide SEQ ID NO:6 + + + + 7. OM-H2276 monoclonal antibody IgG + +
+ + 8. OM-H5831 monoclonal antibody IgG + + + + 9. OMF-H7712 Fab
fragment (Mab) IgG Fab2 + + + + 10. OMF-H6290 Fab fragment (Mab)
IgG Fab2 + + + + 11. XR-178892 fungal extract library natural (u) +
+ + + 12. XR-397344 fungal extract library natural (u) + + + + 13.
XR-573632 fungal extract library natural (u) + + + + 14. SY-73273M
combinatorial library synthetic (u) + + + + 15. SY-32340L
combinatorial library synthetic (u) + + + + 16. SY-95734E
combinatorial library synthetic (u) + + + +
[0050] Corticospinal Tract (CST) Regeneration Assay. High affinity
OMgp binding agents demonstrating inhibition of OMgp-mediated in
vitro axon growth cone collapse as described above are assayed for
their ability to improve corticospinal tract (CST) regeneration
following thoracic spinal cord injury by promoting CST regeneration
into human Schwann cell grafts in the methods of Guest et al.
(1997, supra). For these data, the human grafts are placed to span
a midthoracic spinal cord transection in the adult nude rat, a
xenograft tolerant strain. OMgp binding agents determined to be
effective in in vitro collapse assays are incorporated into a
fibrin glue and placed in the same region. Anterograde tracing from
the motor cortex using the dextran amine tracers, Fluororuby (FR)
and biotinylated dextran amine (BDA), are performed. Thirty-five
days after grafting, the CST response is evaluated qualitatively by
looking for regenerated CST fibers in or beyond grafts and
quantitatively by constructing camera lucida composites to
determine the sprouting index (SI), the position of the maximum
termination density (MTD) rostral to the GFAP-defined host/graft
interface, and the longitudinal spread (LS) of bulbous end
terminals. The latter two measures provide information about axonal
die-back. In control animals (graft only), the CST do not enter the
SC graft and undergo axonal die-back. As shown in Table 3, the
exemplified binding agents dramatically reduce axonal die-back and
cause sprouting and these in vivo data are consistent with the
corresponding growth cone collapsing activity.
3TABLE 3 In Vitro and Vivo Neuronal Regeneration with Exemplary
OMgp Binding Agents. Collapse Reduced OMgp Binding Agent Inhibition
Die-Back Promote Sprouting 1. hNR260/308 + + + + + + + + + + + + 2.
mNR260/308 + + + + + + + + + + + + 3. rNR260/308 + + + + + + + + +
4. s1NR260/308 + + + + + + + + + + + + 5. s2NR260/308 + + + + + + +
+ + 6. s3NR260/308 + + + + + + + + + + + + 7. OM-H2276 + + + + + +
+ + + + + + 8. OM-H5831 + + + + + + + + + + + + 9. OMF-H7712 + + +
+ + + + + + 10. OMF-H6290 + + + + + + + + + 11. XR-178892 + + + + +
+ + + + 12. XR-397344 + + + + + + + + + + + + 13. XR-573632 + + + +
+ + + + + + + + 14. SY-73273M + + + + + + + + + 15. SY-32340L + + +
+ + + + + + + + + 16. SY-95734E + + + + + + + + +
[0051] Peripheral Nerve Regeneration Assay. High affinity OMgp
binding agents demonstrating inhibition of OMgp-mediated in vitro
axon growth cone collapse as described above are also incorporated
in the implantable devices described in U.S. Pat. No. 5,656,605 and
tested for the promotion of in vivo regeneration of peripheral
nerves. Prior to surgery, 18 mm surgical-grade silicon rubber tubes
(I.D. 1.5 mm) are prepared with or without guiding filaments (four
10-0 monofilament nylon) and filled with test compositions
comprising the binding agents of Table 2. Experimental groups
consist of: 1. Guiding tubes plus Biomatrix 1.TM. (Biomedical
Technologies, Inc., Stoughton, Mass.); 2. Guiding tubes plus
Biomatrix plus filaments; 3-23. Guiding tubes plus Biomatrix 1.TM.
plus binding agents.
[0052] The sciatic nerves of rats are sharply transected at
mid-thigh and guide tubes containing the test substances with and
without guiding filaments sutured over distances of approximately 2
mm to the end of the nerves. In each experiment, the other end of
the guide tube is left open. This model simulates a severe nerve
injury in which no contact with the distal end of the nerve is
present. After four weeks, the distance of regeneration of axons
within the guide tube is tested in the surviving animals using a
functional pinch test. In this test, the guide tube is pinched with
fine forceps to mechanically stimulate sensory axons. Testing is
initiated at the distal end of the guide tube and advanced
proximally until muscular contractions are noted in the lightly
anesthetized animal. The distance from the proximal nerve
transection point is the parameter measured. For histological
analysis, the guide tube containing the regenerated nerve is
preserved with a fixative. Cross sections are prepared at a point
approximately 7 mm from the transection site. The diameter of the
regenerated nerve and the number of myelinated axons observable at
this point are used as parameters for comparison.
[0053] Measurements of the distance of nerve regeneration document
therapeutic efficacy. Similarly, plots of the diameter of the
regenerated nerve measured at a distance of 7 mm into the guide
tube as a function of the presence or absence of one or more
binding agents demonstrate a similar therapeutic effect of all 16
tested. No detectable nerve growth is measured at the point sampled
in the guide tube with the matrix-forming material alone. The
presence of guiding filaments plus the matrix-forming material (no
agents) induces only very minimal regeneration at the 7 mm
measurement point, whereas dramatic results, as assessed by the
diameter of the regenerating nerve, are produced by the device
which consisted of the guide tube, guiding filaments and binding
agent compositions. Finally, treatments using guide tubes
comprising either a matrix-forming material alone, or a
matrix-forming material in the presence of guiding filaments,
result in no measured growth of myelinated axons. In contrast,
treatments using a device comprising guide tubes, guiding
filaments, and matrix containing binding agents compositions
consistently result in axon regeneration, with the measured number
of axons being increased markedly by the presence of guiding
filaments.
[0054] OMgp-Specific Monoclonal Antibodies Promote Axon
Regeneration In Vivo. In these experiments, our OM-H2276 and
OM-H5831 OMgp-specific monoclonal antibodies are shown to promote
axonal regeneration in the rat spinal cord. Tumors producing our
OMgp-specific antibodies, implantation protocols and experimental
design are substantially as used for IN-1 as described in Schnell
et al., Nature Jan. 18, 1990; 343(6255):269-72. In brief, our
OM-H2276 and OM-H5831 monoclonal antibodies are applied
intracerebrally to young rats by implanting antibody-producing
tumours. In 2-6-week-old rats we make complete transections of the
corticospinal tract, a major fibre tract of the spinal cord, the
axons of which originate in the motor and sensory neocortex.
Previous studies have shown a complete absence of cortico-spinal
tract regeneration after the first postnatal week in rats, and in
adult hamsters and cats. In our treated rats, significant sprouting
occurs at the lesion site, and fine axons and fascicles can be
observed up to 7-11 mm caudal to the lesion within 2-3 weeks. In
control rats, a similar sprouting reaction occurs, but the maximal
distance of elongation rarely exceeded 1 mm. These results
demonstrate the capacity for CNS axons to regenerate and elongate
within differentiated CNS tissue after neutralization of
OMgp-mediated axon growth inhibtion.
[0055] OMgp-Specific Monoclonal Antibody Fragments Promote Axon
Regeneration in Vivo. In these experiments, OMgp-specific
monoclonal antibody fragments are shown to promote sprouting of
Purkinje cell axons. Experimental protocols were adapted from Buffo
et al., 2000, J Neuroscience 20, 2275-2286.
[0056] Animals and surgical procedures. Adult Wistar rats (Charles
River, Calco, Italy) are deeply anesthetized by means of
intraperitoneal administration of a mixture of ketamine (100 mg/kg,
Ketalar; Bayer, Leverkusen, Germany) and xylazine (5 mg/kg, Rompun;
Bayer).
[0057] Fab fragment or antibody injections are performed as
previously described (Zagrebelsky et al., 1998). Animals are placed
in a stereotaxic apparatus, and the dorsal cerebellar vermis
exposed by drilling a small hole on the posterosuperior aspect of
the occipital bone. The meninges are left intact except for the
small hole produced by the injection pipette penetration. In test
rats a recombinant Fab fragment of the OM-H2276 and OM-H5831
antibodies (produced in E. coli), which neutralizes OMgp-associated
axon growth cone collapse in vitro is injected into the cerebellar
parenchyma. Three 1 .mu.l injections of Fab fragments in saline
solution (5 mg/ml) are performed 0.5-1 mm deep along the cerebellar
midline into the dorsal vermis (lobules V-VII). The injections are
made by means of a glass micropipette connected to a PV800
Pneumatic Picopump (WPI, New Haven, Conn.). The frequency and
duration of pressure pulses are adjusted to inject 1 .mu.l of the
solution during a period of .about.10 min. The pipette is left in
situ for 5 additional minutes to avoid an excessive leakage of the
injected solution. As a control, an affinity-purified F(ab').sub.2
fragment of a mouse anti-human IgG (Jackson ImmunoResearch
Laboratories, West Grove, Pa.) is applied to another set of control
rats using the same procedure. Survival times for these two
experimental sets are 2, 5, 7 and 30 d (four animals for each time
point). An additional set of intact animals are examined as
untreated controls.
[0058] Histological procedures. At different survival times after
surgery, under deep general anesthesia (as above), the rats are
transcardially perfused with 1 ml of 4% paraformaldehyde in 0.12 M
phosphate buffer, pH 7.2-7.4. The brains are immediately dissected,
stored overnight in the same fixative at 4.degree. C., and finally
transferred in 30% sucrose in 0.12 M phosphate buffer at 4.degree.
C. until they sink. The cerebella are cut using a freezing
microtome in several series of 30-.mu.m-thick sagittal sections.
One series is processed for NADPH diaphorase histochemistry. These
sections are incubated for 3-4 hr in darkness at 37.degree. C. in a
solution composed of -NADPH (1 mg/ml, Sigma, St. Louis, Mo.) and
nitroblue tetrazolium (0.2 mg/ml, Sigma) in 0.12 M phosphate buffer
with 0.25% Triton X-100. In some cases (two animals per treated and
control sets at 2 and 5 d survival), microglia are stained by
incubating one section series with biotinylated Griffonia
simplicifolia isolectin B4 [1:100 in phosphate buffer with 0.25%
Triton X-100; Sigma (Rossi et al., 1994a)] overnight at 4.degree.
C. Sections are subsequently incubated for 30 min in the
avidin-biotin-peroxidase complex (Vectastain, ABC Elite kit,
Vector, Burlingame, Calif.) and revealed using the 3,3'
diaminobenzidine (0.03% in Tris HCl) as a chromogen.
[0059] All of the other series are first incubated in 0.3%
H.sub.2O.sub.2 in PBS to quench endogenous peroxidase. Then, they
are incubated for 30 min at room temperature and overnight at
4.degree. C. with different primary antibodies: anti-calbindin
D-28K (monoclonal, 1:5000, Swant, Bellinzona, Switzerland), to
visualize Purkinje cells; anti-c-Jun (polyclonal, 1:1000, Santa
Cruz Biotechnology, Santa Cruz, Calif.); and anti-CD11b/c
(monoclonal OX-42, 1:2000, Cedarlane Laboratories, Hornby, Ontario)
to stain microglia. All of the antibodies are diluted in PBS with
0.25% Triton X-100 added with either normal horse serum or normal
goat serum depending on the species of the second antibody.
Immunohistochemical staining is performed according to the
avidin-biotin-peroxidase method (Vectastain, ABC Elite kit, Vector)
and revealed using the 3,3' diaminobenzidine (0.03% in Tris HCl) as
a chromogen. The reacted sections are mounted on chrome-alum
gelatinized slides, air-dried, dehydrated, and coverslipped.
[0060] Quantitative analysis. Quantification of reactive Purkinje
cells in the different experiments is made by estimating the
neurons labeled by c-Jun antibodies as previously described
(Zagrebelsky et al., 1998). For each animal, three immunolabeled
sections are chosen. Only vermal sections close to the cerebellar
midline that contain the injection sites are considered. The
outline of the selected sections is reproduced using the
Neurolucida software (MicroBrightField, Colchester, Vt.) connected
to an E-800 Nikon microscope, and the position of every
single-labeled cell carefully marked. The number of labeled cells
present in the three reproduced sections is averaged to calculate
values for every individual animal, which are used for statistical
analysis carried out by Student's t test.
[0061] A morphometric analysis of Purkinje axons in the different
experimental conditions for each animal, is performed using three
anti-calbindin-immunolabeled sections, contiguous to those examined
for c-Jun, as described in Buffo et al. (supra). Morphometric
measurements are made on 200.times.250 .mu.m areas of the granular
layer chosen by superimposing a grid of this size on the section.
The selected areas encompass most of the granular layer depth and
contain only minimal portions of Purkinje cell layer or axial white
matter. In each of the selected sections is sampled one area from
the dorsal cortical lobules and one from the ventral cortical
lobules. In addition, to sample from the different parts of these
two cortical regions, areas from different lobules are selected in
the three sections belonging to each individual animal, one area in
each of lobules V, VI, and VII and one in lobules I, II, and IX.
All of the anti-calbindin-immunolabeled Purkinje axon segments
contained within the selected areas are reproduced using the
Neurolucida software (MicroBrightField) connected to an E-800 Nikon
microscope with 20.times. objective, corresponding to 750.times.
magnification on the computer screen. Each labeled axon segment or
branch is reproduced as a single profile. From these reproductions
the software calculates the number of axon profiles, their
individual length, and the total length of all the reproduced
segments, the mean profile length (total length/number of
profiles), and the number of times that the axons crossed a
25.times.25 .mu.m grid superimposed on the selected area. Data
calculated from the different areas in the three sections sampled
from each cerebellum are averaged to obtain values for every
individual animal. Statistical analysis is performed on the latter
values (n=4 for all groups at all time points) by Student's t test
and paired t test.
[0062] Our results reveal significant promotion of sprouting of
Purkinje cell axons in test rats subject to our OM-H2276 and
OM-H5831 OMgp-specific monoclonal antibody fragments as compared
with the control animals.
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[0120] The foregoing descriptions of particular embodiments and
examples are offered by way of illustration and not by way of
limitation. All publications and patent applications cited in this
specification and all references cited therein are herein
incorporated by reference as if each individual publication or
patent application or reference were specifically and individually
indicated to be incorporated by reference. Although the foregoing
invention has been described in some detail by way of illustration
and example for purposes of clarity of understanding, it will be
readily apparent to those of ordinary skill in the art in light of
the teachings of this invention that certain changes and
modifications may be made thereto without departing from the spirit
or scope of the appended claims.
Sequence CWU 1
1
6 1 49 PRT human 1 Asn Pro Trp Val Cys Asp Cys Arg Ala Arg Pro Leu
Trp Ala Trp Leu 1 5 10 15 Gln Lys Phe Arg Gly Ser Ser Ser Glu Val
Pro Cys Ser Leu Pro Gln 20 25 30 Arg Leu Ala Gly Arg Asp Leu Lys
Arg Leu Ala Ala Asn Asp Leu Gln 35 40 45 Gly 2 49 PRT mouse 2 Asn
Pro Trp Val Cys Asp Cys Arg Ala Arg Pro Leu Trp Ala Trp Leu 1 5 10
15 Gln Lys Phe Arg Gly Ser Ser Ser Glu Val Pro Cys Asn Leu Pro Gln
20 25 30 Arg Leu Ala Asp Arg Asp Leu Lys Arg Leu Ala Ala Ser Asp
Leu Glu 35 40 45 Gly 3 49 PRT bovine 3 Asn Pro Trp Val Cys Asp Cys
Arg Ala Arg Pro Leu Trp Ala Trp Leu 1 5 10 15 Gln Lys Phe Arg Gly
Ser Ser Ser Gly Val Pro Ser Asn Leu Pro Gln 20 25 30 Arg Leu Ala
Gly Arg Asp Leu Lys Arg Leu Ala Thr Ser Asp Leu Glu 35 40 45 Gly 4
47 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide sequence 4 Pro Ala Leu Cys Leu Cys Arg Ala Arg
Pro Leu Trp Ala Trp Leu Gln 1 5 10 15 Lys Phe Arg Gly Ser Ser Ser
Glu Val Pro Cys Ser Leu Pro Gln Arg 20 25 30 Leu Ala Gly Arg Asp
Leu Lys Arg Leu Ala Ala Asn Asp Leu Ala 35 40 45 5 40 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide sequence 5 Arg Pro Leu Trp Ala Trp Leu Gln Lys Phe Arg Gly
Ser Ala Ser Glu 1 5 10 15 Val Pro Cys Ser Leu Pro Gln Arg Leu Ala
Gly Arg Asp Leu Lys Arg 20 25 30 Leu Ala Ala Asn Asp Leu Gln Gly 35
40 6 43 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide sequence 6 Gln Pro Ala Val Leu Asp Cys Arg Ala
Arg Pro Leu Trp Ala Trp Leu 1 5 10 15 Gln Lys Phe Arg Gly Ser Ser
Ser Glu Val Pro Leu Ser Leu Pro Gln 20 25 30 Arg Leu Ala Gly Arg
Asp Leu Lys Arg Leu Ala 35 40
* * * * *